The overall objective of this grant is to biochemically and genetically characterize mammalian branched chain alpha-ketoacid dehydrogenase (BCKAD). This is most easily done by purifying the enzyme which is a specific aim of this investigation. A freeze-thaw solubilized extract of beef liver mitochondria is further purified by salt precipitation, CaPO4 gel chromatography and sucrose gradient centrifugation. This preparation has a molecular weight ca 216,000 daltons and shows a typical flavin spectra. The complex will decarboxylate only alpha-keto-isovalerate, alpha-keto-beta-methylvalerate and alpha-ketoisocaproate and also has lipoamide dehydrogenase activity. Further purification by affinity chromatography, isoelectric focusing and ion-exchange chromatography is continuing along with the characterization studies. Antibodies to the complex and its component peptides will be produced in rabbits to aid in studying the enzyme in cultured skin fibroblasts. Maple syrup urine disease is a genetic disorder of man which specifically affects the function of BCKAD. Cultured skin fibroblasts from affected patients show the abnormal enzyme function and can be used to study the disease as well as aid in the understanding of normal physiologic function. Immunohistochemical methods will be used to study the gene products in isolated mitochondrial inner membranes from the cultured cells. This tissue will also be used to study the vitamin responsive form of maple syrup urine disease. Culture media thiamine concentration will be varied and its effect on BCKAD evaluated. Direct assessment of the interaction of thiamine pyrophosphate with BCKAD in the membrane fractions from normal and mutant cells will also be made in an effort to study the stabilizing properties of this cofactor. This composite study with the purified enzyme and the cultured human skin fibroblasts will clarify the biochemistry of the BCKAD complex and provide a rationale for management of patients with the disease.